The cardiac natriuretic peptides (NP) atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) are polypeptide hormones synthesized, stored and released by cardiac muscle cells (cardiocytes). In many ways, the endocrine heart is a modulator of systems such as the sympathetic nervous system, the renin-angiotensin-aldosterone system and other determinants of vascular tone, extracellular fluid volume and renal function.
ANF and BNP are synthesized by cardiocytes as preprohormones that are enzymatically processed to yield prohormones and, ultimately, hormones that are released into the circulation. In humans, the prohormone proANF is a polypeptide that contains 126 amino acids (ANF.sub.1-26) that is processed to ANF.sub.1-98 and ANF.sub.99-126, the latter being the biologically active portion. Human proBNP, on the other hand, is 108 amino acids long and it is processed to BNP.sub.1-76 and BNP.sub.77-108, the latter being the biologically active peptide. Both the C-terminal and N-terminal portions of NPs circulate in blood. For example, Hunt et al (1995, Biochem Biophys Res Comm 214:1175-1183) report the detection of hBNP and N-terminal ProBNP within human plasma, and note an increase in the plasma levels of both peptides with congestive heart failure.
Under certain pathophysiological conditions affecting the cardiovascular system, synthesis and release of both ANF and BNP are significantly augmented in both atrial and ventricular cardiocytes. Increased production of ANF and BNP by the mammalian ventricle is a hallmark of cardiac hypertrophy and failure (Vokota, N. et al Am J Hypertens 1995;8:301-310). Further, it is now known that measurement of the circulating levels of different fragments of these hormones in plasma is a powerful means to identify elderly subjects at risk of heart failure (Davis KM. et al. JAAM 1992;267:2625-2629), establish long term prognosis after (myocardial infarction) MI (Hall C. et al J Am Coll Cardiol 1995;26(6):1452-1456), stratify patients in terms of response to angiotensin-converting enzyme inhibition post MI (Motwani JG. et al Lancet 1993;341:1109-1113) and to demonstrate asymptomatic left ventricular dysfunction (Lerman A, Jr. et al Lancet 1993;341:1105-1109; Arad M et al Cardiology 1996;87:12-17).
Myocarditis is also characterized by an increase in synthesis and release of NP from the heart (Takemura G. et al. Int J Cardiol 1995;52:213-22:). While the biological basis of increased production of NP during cardiac hypertrophy and failure is conceptually placed within the re-expression of the cardiac fetal phenotype seen with chronic hemodynamic overload and heart failure, the basis for increased production of NP in myocarditis is not understood. Increased ventricular gene expression in intact cardiocytes surrounding foci of degenerative changes or necrosis has been observed in both animals models and human myocarditis. Mice inoculated with encephalomyocarditis virus, a model of myocarditis with heart failure, showed significantly increased ANF plasma levels and ventricular ANF mRNA 10 and 30 days after infection when compared to non-infected controls (Kanda T. et al. J Pharmacol & Exptl Ther 1995;274:494-498). Treatment of the mice 24 h after inoculation with a combination of the immunomodulators OK432 and human interferon-.alpha. A/D prevented the development of cardiomyopathy and hypertrophy and down regulated the expression of ANF mRNA in the ventricles to near normal levels.
W097/32900 (Mischak RP. et al, published Sep. 12, 1997; which is incorporated by reference) discloses monospecific antibodies to hBNP and their use as diagnostic reagents for the detection of BNP levels in plasma of patients with congestive heart disease. The specific epitopes of the MAb's include fragments of the mature BNP (BNP.sub.77-108) peptide. These fragments include: 5-13 hBNP, 1-10 hBNP, 15-25 hBNP, and 27-32 hBNP.
EP542255 (Tsuji T. et al. published May 19, 1993; which is incorporated by reference) discloses monoclonal antibodies that recognize the C-terminus of hBNP, and the use of these MAb's within RIA's. The assay involves the determination of hBNP in plasma and can be used for the diagnosis of diseases such as hypertension and altered states of the heart and kidney.
Ationu et al (1993a, Cardiovas Res. 27:2135-2139) disclose the monitoring of circulating BNP and ANF levels in paediatric cardiac transplant recipients. In this study increased plasma BNP and ANF levels were noted within patients during the first year after transplant. When BNP levels were re-assayed at 2.5 or 3 years following transplantation, the levels were reduced. Another study (Geny B. et al 1988, J Thorac Cardiovas Surgery 115:473-475) considered the relationship between BNP levels before and immediately following heart transplantation, or coronary artery bypass grafting, and concluded that no meaningful relationship was present. Rather it was observed that following transplantation, plasma BNP levels, which are typically elevated, decreased and returned to earlier levels. Ationu et al (1993b, Cardiovas Res. 27:188-191) disclose the assessment of plasma BNP levels following heart transplantation. Levels of BNP were noted to increase following transplantation, however, there no relationship was observed within plasma or ventricular BNP levels, and rejection episodes. These authors report the desire to derive a non-invasive marker in order to monitor such situations, however, no such relationship was noted with either of these NP's. A similar lack of correlation has been noted in several studies examining circulating ANF levels following cardiac transplantation (e.g. Masters RG. et al Can J. Cardiol 1993,9:609-617).
In all of these studies, there is no disclosure of a relationship between circulating BNP levels and transplant rejection, nor is there any demonstration that circulating BNP levels can be used to monitor cardiac allograph rejection. Rather, several of these references demonstrate the lack of such a correlation, and lack of utility of BNP levels as an indicator of rejection. Even though the prior art has failed to report any meaningful relationship between levels of BNP in biological fluids and transplant rejection, the present invention demonstrates such a relationship, and provides a method for the detection of myocardial allograph rejection by determining BNP levels within biological fluids.